An illustration showing the structure of a cell-signaling cytokine called erythropoietin (EPO). It has long been thought that when EPO binds with its receptor, EPOR, it functions like an on/off switch, triggering red blood cell production. New findings suggest that this process is more nuanced than previously thought; even slight variations to cytokines like EPO can cause disease.

While researching a rare blood disorder called Diamond-Blackfan anemia, scientists stumbled upon an even rarer anemia caused by a previously-unknown genetic mutation. During their investigation, the team of scientists — from the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, the Broad Institute of Harvard and MIT and Yale University — had the relatively unusual opportunity to develop an “on-the-fly” therapy.

As they analyzed the genes of one boy who had died from the newly-discovered blood disorder, the team’s findings allowed them to help save the life of his infant sister, who was also born with the same genetic mutation. The results were recently reported in Cell.

“We had a unique opportunity here to do research, and turn it back to a patient right away,” says Vijay Sankaran, MD, PhD, the paper’s co-corresponding author and a principal investigator at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center. “It’s incredibly rewarding to be able to bring research full circle to impact a patient’s life.”

Expanding our understanding of cytokines

For a long time, it was thought that small proteins called cytokines, which are responsible for signaling cell behavior, functioned like on/off switches. As long as the signal was flipped on — no matter how or for how long — it should activate the relevant cell behavior.

But now, a molecular investigation into the genomes of these two siblings has led to the discovery that cytokine signals are actually quite finely tuned by nature. As it turns out, changes to that tuning can result in human disease, such as this new, very rare kind of anemia.

Anemia occurs when the body can’t produce enough red blood cells, which are vitally important because they contain an oxygen-transporting protein called hemoglobin.

“What’s really excited us about this work is that — on a very basic science level — it’s made us realize we can tune the activity of a lot of different cytokines,” says Sankaran. “That knowledge has important therapeutic implications.”

Finding a needle in a haystack

Analyzing cases from around the world, the team was toiling away at uncovering new genetic underpinnings related to Diamond-Blackfan anemia. The rare disorder, first described over 80 years ago by physicians including Louis Diamond and Kenneth Blackfan of Boston Children’s Hospital, impairs red blood cell production due to an absence of proper precursors in the bone marrow. Soon, Sankaran’s team found themselves amidst a baffling case.

A 6-year-old boy had failed to respond to therapies that are normally effective at treating Diamond-Blackfan anemia, including a stem cell transplant from his maternal aunt. She was considered a perfect match for him, yet, he passed away five months after the transplant.

Even more curious, Diamond-Blackfan anemia is usually considered to show autosomal dominant inheritance, which means that a parent has a 50 percent chance of passing the disease to their offspring. But neither of the boy’s parents, who were first cousins, had the disorder.

So, the team performed sequencing of the boy’s exome, analyzing all of the genes expressed in his genome. Instead of finding mutations known to be associated with Diamond-Blackfan, they pinpointed a previously-unknown mutation in his EPO gene. EPO codes for production of the cytokine erythropoietin (EPO), which has the subsequent job of stimulating red blood cell creation. This particular genetic mutation, called EPO R150Q, had resulted in a switch of amino acids at position 150 on EPO.

Binding speed matters

To understand the mutation’s effect, Sankaran’s team did tests comparing the function of normal EPO to a replica of the boy’s mutant EPO. They discovered, interestingly, that the mutation didn’t alter EPO’s ability to bind to its receptor (EPOR), but that it did alter the rate in which it attached and detached.

“Normally, EPO attaches to EPOR for a matter of minutes,” says Sankaran. “But in this case, we saw it attaching only for a matter of seconds.”

This, apparently, was not a long enough connection to spur red blood cell production. Even though the boy had enough of his own EPO circulating in his body, the mutated rate and speed with which it attached to EPOR prevented him from making enough red blood cells.

The EPO R150Q mutation results in a switched amino acid at position 150 on the erythropoietin (green) structure. This location plays an important role in binding EPO with its receptor (aqua).

“We’re seeing that the kinetics of cytokine signaling matter very much when it comes to downstream effects,” says Sankaran. “This is very important clinically, and it opens up the possibility to fine tune therapies that, so far, haven’t worked exactly the way we wanted them to. By changing the kinetics of certain cytokine-receptor interactions, we might be able to more precisely stimulate some activities, and not others.”

Turning it back to the patient

The little boy, whose unique anemia caused Sankaran’s team to discover the nuances of cytokine signaling, still had one more gift to give.

As Sankaran’s team was working on this paper, the boy’s parents gave birth to a daughter. Amazingly, she too was born with severe anemia and possessed the same genetic mutation, EPO R150Q.

Fresh off their investigations into EPO R150Q, Sankaran’s team recommended that she be given a trial treatment of recombinant EPO therapy. This synthetically-made EPO hormone acts just like normal, natural EPO, and is often prescribed for people experiencing anemia due to chemotherapy. Therefore, the team hypothesized that recombinant EPO could help her body activate production of red blood cells.

Recombinant EPO is often used by cancer patients, whose red blood cells are depleted by chemotherapy. The drug has also been used controversially by elite athletes. Since red blood cells carry hemoglobin, a protein that transports oxygen, athletes who take EPO may have a competitive advantage.

With consent from her family, she was given recombinant EPO. Within the first 11 weeks, she showed a strong response to the therapy and her production of red blood cells increased. This robust drug response characterizes her and her late brother’s rare anemia as a distinct, treatable blood disorder.

“This is true precision medicine,” says Sankaran. “We have been able to positively alter the course of a patient’s life, which is exactly what research efforts at a children’s hospital are meant to do.”